The investigation on different functions of the GTP-binding protein Rho1p in yeast has continued. In previous work it had been found that Rho1p is required for activity and regulation of beta(1-3)glucan synthase and for activation of protein kinase C. In the present period, a function of Rho1 in cell polarization has been explored. Some mutants in the switch 1 domain of Rho1p, one of two regions that change conformation upon GTP binding, are temperature-sensitive. It has been found that such mutants, in particular rho1E45I, are blocked at an early stage of the cell cycle at the nonpermissive temperature (37oC). The mutant cells fail to bud and become round and large. Actin filaments do not polarize to the presumptive bud site. Other proteins, such as Cdc42p and Spa2p, that normally localize to the same site independently from actin, also remain delocalized. In contrast to cdc42 mutants, that show a similar morphology, nuclei of rho1E45I do not divide, although duplication of DNA takes place slowly at 37oC. Thus, it would appear that the execution of Rho1p function is previous to that of Cdc42p. The polarization defect of rho1 mutants extends also to mating. The mutants fail to form mating projections and to recruit actin to the projections in the presence of mating pheromone. Other experiments excluded that the mutant defects were due to impairment of the Rho1p functions in glucan synthesis or protein kinase C activation. These results show that Rho1p is essential for cell polarization. Experiments with another temperature-sensitive mutant of Rho1p, rho1-104, showed that high concentrations of sorbitol in the growth medium suppressed almost completely the growth defect at the nonpermissive temperature. Both the amount of protein and its activity in the glucan synthase system were preserved by sorbitol. It is known that high concentrations of osmolytes in the medium induce accumulation of glycerol in the yeast cell. This was also the case in our strain. Introduction of a mutation in a glycerol phosphate dehydrogenase in the rho1-104 mutant resulted in decreased accumulation of glycerol and also in decreased suppression of the defect at 37oC. It was concluded that glycerol, a known protein stabilizer, was the protector of Rho1p, when the medium contained high concentrations of sorbitol. This result explains why high concentrations of osmolyte protect many mutants (osmotic-remedial mutants), even when the mutation is not related to cell wall synthesis or cell integrity.